Revascularization is a key determining factor in bone graft healing and repair. Autografts are vastly superior to allografts and synthetic bone grafts largely due to the fact that autografts can be rapidly revascularized and form new bone whereas allografts cannot. While vascularization of bone tissue has been increasingly recognized as a key factor in repair and reconstruction, our understanding of bone graft vascularization in bone transplantation has been limited to histological observations described in the early 70s and 80s. These descriptions are often restricted by dependency on histology which prohibits three-dimensional and spatiotemporal analyses of vascularization of the grafted bone. We have recently established a cranial bone window chamber model which allows high resolution, four-dimensional imaging and analyses of bone defect healing over a period of months using multiphoton laser scanning microscopy (MPLSM). By transplanting allograft and autograft bone into this windowed bone defect model, we were able to track the revascularization process and demonstrate the fundamental differences between allografts and autografts in living animals. The goal of our current proposal is to utilize this novel intravital imaging approach combined with transgenic animal models to gain a better understanding of the vascularization mechanisms of bone graft transplantation. Based on our preliminary date and recent literature on the key role of hypoxia-inducible factor 1- alpha (HIF-1?) in oxygen sensing and coupling of osteogenesis and angiogenesis, two complementary Aims are proposed. Aim 1 will examine the key role of the HIF-1 pathway in revascularization and repair by transplantation of a HIF-1 deficient or over-activated live bone isograft into a cranial defect window chamber model. Aim 2 will determine the effects of engraftment of MSCs with enhanced HIF-1 signaling on bone allograft revascularization and repair. A novel oxygen sensor, which allows quantitative measurements of oxygen tension simultaneously with osteogenesis and angiogenesis in vivo will be established. The completion of our current project will enhance our knowledge of graft healing and revascularization and further offer rationales and strategies to augment the efficacy of future cell-based therapy aimed at enhancing bone repair and regeneration. Understanding the complex role of hypoxia and its master regulators in bone graft revascularization and bone healing will further aid in the development of novel pharmaceutical agents that can redress the detrimental outcomes often seen in repair and scarring of bone allograft healing.